Nickel sulphide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards, well documented and acceptable for publication.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

publication

Title:

Genotoxic activity of nickel subsulphide alpha-Ni3S2

Author:

Arrouijal FZ, Hildebrand HF, VophiI H, Marzin D

Year:

1990

Bibliographic source:

Mutagenesis. 5(6):583-589

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The micronucleus test was carried out in the OF1 strain of mice after receiving a single i.p. injection of Ni3S2 suspended in 1% carboxymethyl cellulose.

GLP compliance:

not specified

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Trinickel disulphide

EC Number:

234-829-6

EC Name:

Trinickel disulphide

Cas Number:

12035-72-2

Molecular formula:

Ni3S2

IUPAC Name:

Trinickel disulphide

Details on test material:

- Name of test material (as cited in study report): alpha-Ni3S2, nickel subsulphide
- Analytical purity: 99.5%
- Other: particle size < 2um

Test animals

Species:

mouse

Strain:

other: OF1 strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: lffa Credo, L' Arbresle, France
- Age at study initiation: 8 wk
- Weight at study initiation: 20 +/- 2 g

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: 1 % carboxymethyl cellulose

Details on exposure:

Not oral, dermal, or inhalation.

Duration of treatment / exposure:

acute

Frequency of treatment:

once

Post exposure period:

24-72 hr

Doses / concentrations

Remarks:

Doses / Concentrations:
0.25 g Ni3S2/kg
Basis:
nominal conc.

No. of animals per sex per dose:

5 males, 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: i.p.
- Doses / concentrations: 50 mg/kg

Examinations

Tissues and cell types examined:

femoral bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: 100% survival; lethality at higher doses

DETAILS OF SLIDE PREPARATION & ANALYSIS:
Bone marrow from treated and vehicle control animals were collected in FCS. Cells were spread on coded slides and stained with the May-Grunwald/Giemsa technique.

Evaluation criteria:

A total of 1000 polychromatic erythrocytes per animal were examined for micronuclei and the polychromatic erythrocytes rate was determined on 200 cells.

Statistics:

Statistical evaluation of the results was performed by using the Mann-Whitney U-test.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Evidence of cytotoxicity in tissue analyzed: reduction in polychromatic erythrocytes (PCEs) - see Table
- Rationale for exposure: 100% survival
- Harvest times: 24, 48, 72 hr

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): significant (see Table)
- Ratio of PCE/NCE (for Micronucleus assay): 71% in control, 44 % in Ni3S2 treated at 24 hr
- Statistical evaluation: see table

Any other information on results incl. tables

Micronucleus Test in Mice with i.p. Ni3S2

	% PCEs (SE)			No. micronuclei (SE)
	24 hr	48 hr	72 hr	24 hr	48 hr	72 hr
Control	71.1 (7.8)	61 (8.5)	57 (8.0)	0.5 (0.7)	0.1 (0.3)	0.4 (0.5)
Ni3S2, 250 mg/kg	43.5 (7.8)***	45.7 (11.8)**	43.4 (13.0)	3.4 (1.1)**	2.1 (1.3)**	1.5 (0.9)*
Cyclophosphamide, 50 mg/kg	70.8 (7.4)	ND	ND	46.4 (7.3)**	ND	ND

*, P < 0.05

**, P < 0.01

***, P < 0.001

ND, not done

Toxicity and Bone Marrow Ni3S2 Levels in Mice
Dose (mg/kg)	Survival (%) After 72 hr	Mean ug Ni/10^6 Cells in Bone Marrow (SE)
Control	100	1.2 (0.4)
Ni3S2....250	100	4.8 (1.9)*
..............500	83	6.7 (2.6)*
.............. 1000	67	10.8 (1.7)***
..............2000	34	13.6 (4.2)**

*, P < 0.05

**, P < 0.01

***, P < 0.001

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): positive
Together with in vitro findings discussed elsewhere (section 7.6.1 Genetic Toxicity In Vitro of nickel subsulphide IUCLID file), the authors concluded that Ni3S2 can cause in vivo clastogenic effects - as shown in bone marrow cells. This study also showed that i.p. injection of Ni3S2 resulted in elevated Ni levels in the bone marrow.

Executive summary:

Arrouijal et al. (1990) examined the clastogenic and mutagenic effects of Ni3S2 using in vitro and in vivo systems (in vitro results are described in the Genetic Toxicity In Vitro Section). In this study, the micronucleus test was carried out in the OF1 strain of mice that received a single i.p. injection of Ni3S2 suspended in 1% carboxymethyl cellulose. A prior experiment with 0.25, 0.5 and 1 mg/kg determined that 100% survival occurred only at the lower dose, and was thus chosen for the micronucleus test. For the micronucleus test, control and treated animals were sacrificed at 24, 48, and 72 hours after exposure (10 animals per group) and the femoral bone marrow cells were harvested. Animals were exposed to 50 mg/kg cyclophosphamide as a positive control. Relative to controls, these animals exhibited no change in polychromatic erythrocytes (PCEs), but a significant increase in micronuclei per 1000 PCEs (0.5 ± 0.7 vs 46.4 ± 7.3). Relative to control animals, Ni3S2 treated animals exhibited a significant decrease in PCEs at 24 and 48 hours after treatment, which the authors noted as evidence for bone marrow toxicity. PCEs comprised roughly 71% of 200 bone marrow cells evaluated in control and cyclophosphamide treated animals, and only 44% in Ni3S2 treated animals. The number of micronuclei in PCEs from treated animals was significantly elevated at 24 (3.4 ± 1.1), 48 (2.1 ± 1.3), and 72 hr (1.5 ± 0.9) hr after exposure. Together with in vitro findings discussed elsewhere (see above), the authors concluded that Ni3S2 can cause in vivo clastogenic effects. This study also presented Ni content data in femoral bone marrow cells taken from animals dosed with 0.25, 0.5, 1 or 2 mg/kg Ni3S2 by i.p. The 3-day survival and Ni levels are as follows: 0.25 mg/kg (100%, 4.8 ± 1.9), 0.5 mg/kg (83%, 6.7 ± 2.6), 1 mg/kg (67%, 10.8 ± 1.7), and 2 mg/kg (34%, 13.6 ± 4.2). These findings demonstrate that i.p. injection of suspended Ni3S2 at levels at or above 250 mg/kg can result in Ni reaching the bone marrow, and that Ni3S2 can cause clastogenic effects in vivo. STUDY RATED BY AN INDEPENDENT REVIEWER